In previous studies conducted as a postdoctoral fellow in Dr. David Beach's laboratory at Cold Spring Harbor Laboratory, the P.I. discovered that cyclin-CDK complexes are associated with a number of small cellular proteins, including p2l and pl6. P2l (also known as CIPI, WAF1, SDII, CAP20, PICI, CDKNI) regulates the cell cycle through at least three different pathways: p2l encodes an potent inhibitor of all cyclin CDK enzymes that have been tested, p2l protein interacts with PCNA independently of cyclin-CDK enzymes to inhibit replicative DNA synthesis, and p2l can also regulate the activity of the E2F transcription factor via its interaction with cyclin-CDK proteins. Transcription of p21 is stimulated by the tumor suppressor p53, and therefore this regulation provides a direct link between tumor suppression by p53 and cell cycle control. pl6 (also known as INK4, MTSI, CDK4I, CDKN2) encodes a cyclin D- CDK4 specific inhibitor and is deleted or mutated in a wide variety of human tumor-derived cell lines and in several specific types of primary tumors. Very recently, the P.I.'s laboratory identified at least four new CDK4- and CDK6-interacting small cellular proteins (pl4, pl5, pl8 and p20) and have isolated and functional characterized the gene encoding two of these four proteins, pl8, and p14MTS2. pl8 and pl4MTS2 are related to pl6 in sequence, function and evolution, and thus they define a new and potentially large family of CDK inhibitors. The four specific aims of this proposal are concerned with the pl6/pl8 family of CbK inhibitors that controls cell growth by regulating the activity of the retinoblastoma gene product, pRb, and that is strongly implicated in the development of human cancer. Specific Aim I. Identification and Cloning of Novel CDK4- and CDK6- Associated Cellular Proteins. Experiments proposed in this section are aimed at identification and cloning of genes encoding novel CDK4- and CDK6-associated proteins including pl5 and p20. Specific Aim II. Characterization of Novel CDK4- and CDK6-Associated Cellular Proteins. Experiments proposed in this section aim at the initial characterization of genes encoding CDK4-and CDK6-associated proteins isolated by the experiments proposed in Specific Aim I. Specific Aim III. Mechanism of pl8 and pl6 Function. The experiments in this section are aimed at (A) Demonstration in vivo of a growth suppression pathway involving pl6/pl8. (B) Characterization of pRb- and p53-mediated repression of pl6 transcription. ~ Elucidation of the pl6/pl8 inhibitory mechanism. (D) Investigation of p18 function in muscle cell differentiation. Specific Aim IV. Searching for p18 and p 14MTS2 mutations. In this section, we propose experiments to search for p18 and MTS2 gene mutations t investigate p18 tumor suppression function.